Elsevier

Experimental Neurology

Volume 289, March 2017, Pages 117-127
Experimental Neurology

Research Paper
Alterations in hypoglossal motor neurons due to GAD67 and VGAT deficiency in mice

https://doi.org/10.1016/j.expneurol.2016.12.004Get rights and content

Highlights

  • XII motor neurons are responsible for coordinated breathing and sucking at birth.

  • These behaviours are modulated by excitatory and inhibitory inputs onto XII MNs.

  • This loss triggers increased complexity of XII MN’s dendritic arbor and spine density.

  • GABA and Glycine neurotransmitters are key regulators of XII MN development.

Abstract

There is an emerging body of evidence that glycinergic and GABAergic synaptic inputs onto motor neurons (MNs) help regulate the final number of MNs and axonal muscle innervation patterns. Using mutant glutamate decarboxylase 67 (GAD67) and vesicular inhibitory amino acid transporter (VGAT) deficient mice, we describe the effect that deficiencies of presynaptic GABAergic and/or glycinergic release have on the post-synaptic somato-dendritic structure of motor neurons, and the development of excitatory and inhibitory synaptic inputs to MNs. We use whole-cell patch clamp recording of synaptic currents in E18.5 hypoglossal MNs from brainstem slices, combined with dye-filling of these recorded cells with Neurobiotin™, high-resolution confocal imaging and 3-dimensional reconstructions. Hypoglossal MNs from GAD67- and VGAT-deficient mice display decreased inhibitory neurotransmission and increased excitatory synaptic inputs. These changes are associated with increased dendritic arbor length, increased complexity of dendritic branching, and increased density of spiny processes. Our results show that presynaptic release of inhibitory amino acid neurotransmitters are potent regulators of hypoglossal MN morphology and key regulators of synaptic inputs during this critical developmental time point.

Introduction

During development, the mammalian neuromotor system undergoes a period of programmed motor neuron (MN) cell death. Over 50% of all motor neurons generated during neurogenesis are lost in the final trimester in utero (Lance-Jones, 1982, Oppenheim, 1991). The final number of surviving MNs is regulated in an activity-dependent manner, as reduced skeletal muscle activity causes increased MN survival and neuromuscular innervation (Landmesser, 1992, Banks et al., 2003), whereas increased skeletal muscle activity causes decreased MN survival and neuromuscular innervation (Oppenheim and Nunez, 1982). These alterations in muscle activity depend on nervous system activity, that is regulation of MN firing by other central neurons (neuronal activity), which is then passed onto muscle via neuromuscular synapses (Banks and Noakes, 2002).

Glycine and GABA are important inhibitory neurotransmitters in the adult nervous system, and they are vital for normal postnatal neuromotor function and development, as GAD67 and VGAT-deficient mice die within a few hours after birth from respiratory complications and failure to suckle (Asada et al., 1997, Wojcik et al., 2006, Kakizaki et al., 2015). GAD67 is the major synthesizing enzyme for GABA in prenatal and neonatal mice, and electrophysiological studies have shown that GABAergic neurotransmission is reduced or nearly eliminated in brainstem respiratory neurons from GAD67 mutants (Fujii et al., 2007). VGAT is the transporter protein which transfers both GABA and glycine into presynaptic vesicles, and both GABAergic and glycinergic neurotransmission are absent or severely reduced in spinal cord neurons (Wojcik et al., 2006), brainstem respiratory neurons (Fujii et al., 2007), spinal MNs (Saito et al., 2010) and hypoglossal MNs (Rahman et al., 2015) of VGAT mutants.

However, during neural development, GABA and glycine neurotransmission are thought to provide important synaptic sources of depolarizing chloride ion (Cl) conductances to MNs (Nishimaru et al., 1996, Singer and Berger, 2000, Ben-Ari, 2002). This response is due to the high intracellular Cl concentration in foetal and neonatal neurons causing membrane depolarization as Cl exits to the extracellular space when Cl permeable channels gated by glycine and GABA are activated. Closer to birth, or during early postnatal life, the action of GABA and glycine shifts from depolarizing to hyperpolarizing or inhibitory as the intracellular Cl concentration decreases, resulting in movement of Cl into the intracellular compartment upon activation by glycine and/or GABA (Akerman and Cline, 2007), leading to a refinement of the MN inputs to target skeletal muscle. Morphological and physiological studies of mice lacking the glycine receptor-clustering molecule Gephyrin (Kneussel et al., 1999) support this mechanism, as loss of glycinergic neurotransmission leads to increased MN activity and decreased MN survival when compared to wild-type controls (Banks et al., 2005), suggesting that the effect of GABAergic and glycinergic neurotransmission onto hypoglossal MNs is inhibitory at birth.

However, the mechanisms by which increased MN activity leads to greater MN loss, compared to wild-type MNs (Banks et al., 2005, Fogarty et al., 2013b, Fogarty et al., 2015b) remains unclear. One mechanism underlying increased MN loss may be the effects of an imbalance between excitatory and inhibitory inputs onto mutant MNs overloading embryonic MNs with glutamate, a known mediator of excitotoxic neuronal death (Choi, 1992, Choi, 1995), and of dendritic growth (Kalb, 1994, Inglis et al., 2002, Metzger, 2010, Koleske, 2013), and increased motor neuron excitability (van Zundert et al., 2008). By quantifying synaptic activity and morphology of individual MNs from mice with deficiencies in GABA and/or glycinergic neurotransmission, we may thus gain an understanding of how altered central synaptic inputs onto MNs regulate final hypoglossal MN numbers at birth, when the neuromotor system must be functional at birth for suckling and respiration.

This study therefore aimed to quantify alterations to the dendritic arbor, spine density, and functional synaptic inputs of hypoglossal MNs at E18.5, using GAD67- and VGAT-deficient mice, compared to wild-type (WT) MNs. We used patch-clamp electrophysiology techniques to record synaptic activity, followed by dye-filling of the recorded MNs with Neurobiotin™ for subsequent high resolution morphometric measurements. We also confirm our previous qualitative observations of decreased IPSC frequency and increased EPSC frequency of GAD67-deficicient XI MNs and increased somatic spine density in VGAT XII MNs compared to WT controls (Kanjhan et al., 2016b).

Section snippets

Animals used and ethical statement

We used five GAD67-deficient mice (GAD67-/-), seven VGAT-deficient mice (VGAT-/-) and nine wild-type mice (WT) at embryonic day 18/postnatal day 0 (termed E18.5). GAD67-/- and VGAT-/- mice and their respective WT littermates were generated and genotyped in accordance with previous studies (Tamamaki et al., 2003, Saito et al., 2010). All mice including WT littermates were derived from heterozygote GAD67+/- or heterozygote VGAT+/- breeding scheme on a C57Bl/6J genetic background. This embryonic

Decreased frequency of spontaneous inhibitory neurotransmission is coupled with increased frequency of spontaneous excitatory neurotransmission in GAD67- and VGAT-deficient hypoglossal MNs

To investigate the functional changes in inhibitory and excitatory synaptic inputs, we recorded spontaneous inhibitory post-synaptic currents (IPSCs) at 0 mV and spontaneous excitatory post-synaptic currents (EPSCs) at − 60 mV (Kanjhan and Bellingham, 2013, Fogarty et al., 2013a). As anticipated, in hypoglossal MNs from GAD67- and VGAT-deficient mice, which lack GABAergic and GABA/glycinergic neurotransmission respectively, IPSC frequency was significantly decreased, compared to WT MNs (compare

Discussion

Our findings indicate that the presynaptic release of GABA and glycine must play a significant role in regulating functional and morphological changes in MNs during development. GAD67- and VGAT-deficient mice, which lack GABAergic or glycinergic and GABAergic neurotransmission, exhibited increased dendritic length, dendritic complexity, increased somatic and dendritic spiny processes, and increased glutamatergic excitatory synaptic neurotransmission. This is consistent with our observations of

Competing interests

All authors ascertain no conflict of interests associated with this work.

Contributions

R.K., M.J.F., Y.Y., M.C.B. and P.G.N. designed research; R.K. and M.J.F. performed research; M.J.F., R,K., M.C.B. and P.G.N. analyzed data; M.J.F., R,K., M.C.B. and P.G.N. wrote the paper.

Funding

The study was supported by grants to P.G.N. and M.C.B. (National Health and Medical Research Council Project Grants 1065884 and 401579; Motor Neuron Disease Research Institute Australia grant numbers GIA1641, GIA1547 and Y.Y. (Grant-in Aid for Scientific Research on Innovative Areas “Adaptive circuit shift” and Scientific Research (B) from MEXT (grant Nos, 26290002, 15H01415, and 15H05872), and Takeda Science Foundation, Japan ). M.J.F. is supported by an National Health and Medical Research

Acknowledgements

We thank K. Obata for his intellectual and practical support of this project; M. Shayegh and M. White for genotyping; L. Hammond for microscopy assistance (ARC LIEF grant LE100100074). The authors declare no conflict of interest.

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    1

    M.J.F and R.K contributed equally to this study.

    2

    P.G.N and M.C.B contributed equally to this study.

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